CN112063571A

CN112063571A - Engineering bacterium for high yield of L-amino acid and construction method and application thereof

Info

Publication number: CN112063571A
Application number: CN202010821272.3A
Authority: CN
Inventors: 胡丹; 邹德勇; 王成; 薛婷莉; 李岩
Original assignee: Langfang Meihua Bio Technology Development Co Ltd
Current assignee: Langfang Meihua Bio Technology Development Co Ltd
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2020-12-11
Anticipated expiration: 2040-08-14
Also published as: CN112063571B

Abstract

The invention provides an engineering bacterium for high yield of L-amino acid and a construction method and application thereof. The engineering bacteria are gene weakening strains obtained by weakening genes NCgl2493 and/or NCgl0224 in original strains; wherein the original strain is a coryneform L-amino acid-producing bacterium. The engineering bacteria for high yield of L-amino acid provided by the invention have higher L-amino acid (especially L-lysine) production capacity, and NCgl2493 gene and/or NCgl0224 gene expression protein in corynebacteria cells is inactivated or weakened. Experiments show that the engineering bacteria for high yield of L-amino acid is taken as a fermentation strain, the L-lysine yield and the conversion rate are obviously improved, a foundation is laid for the industrial production of L-lysine, and the application prospect is wide.

Description

Engineering bacterium for high yield of L-amino acid and construction method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an engineering bacterium for high yield of L-amino acid, a construction method and application thereof.

Background

L-amino acids, in particular L-lysine, L-threonine and the like, are widely used in the animal feed, pharmaceutical and food industries. Wherein about 90% of L-lysine is used in feed industry, and 10% is used in food and pharmaceutical industry. The L-lysine can be used as animal feed additive, and can help organism to absorb other amino acids, thereby improving feed quality. L-threonine is widely applied to feed, food additives, preparation of auxiliary materials of medicines and the like, is an important nutrition enhancer, can enhance grains, cakes and dairy products, and has the effects of relieving human fatigue and promoting growth and development like tryptophan.

The method for producing the L-amino acid by the microbial fermentation method has the advantages of low raw material cost, mild reaction conditions, easy realization of large-scale production and the like, and is the most important method for producing the L-amino acid at present. Metabolic engineering has improved strain performance to a large extent in recent years, but there remains the potential to continue to improve yield and conversion. Bacteria of the genus Corynebacterium, in particular Corynebacterium glutamicum (Corynebacterium glutamicum), are "generally regarded as safe" (GRAS) strains which are widely used for the production of L-amino acids, in particular L-lysine. Compared with Escherichia coli, the lysine synthesis path of Corynebacterium glutamicum has obvious advantages, and the synthesis of the lysine precursor alpha-diaminopimelic acid only needs one-step enzyme catalytic reaction, namely the diaminopimelic acid dehydrogenase catalysis is completed, while Escherichia coli needs four-step enzyme catalytic reaction. Therefore, the use of Corynebacterium glutamicum for the production of L-lysine has significant advantages.

Corynebacterium glutamicum can likewise be used for the production of L-threonine and has metabolic advantages over E.coli. The reason for this is that Corynebacterium glutamicum has pyruvate carboxylase and can synthesize L-threonine by making full use of pyruvate precursors. The enzyme does not exist in escherichia coli, redundant pyruvic acid generated by glycolysis can only enter TCA circulation and cannot be used for synthesizing L-threonine, and the saccharic acid conversion rate is reduced.

Disclosure of Invention

The invention aims to provide an engineering bacterium for high yield of L-amino acid and a construction method and application thereof.

Another object of the present invention is to provide a method for producing an L-amino acid by fermentation or a method for increasing the fermentation yield of an L-amino acid.

The invention has the following conception: the inventors have, after long-term research and practice, discovered, by chance, that the modification of two genes encoding "hypothetical proteins" (annotated by bioinformatic methods) in the chromosome of coryneform bacteria can contribute to the increased production of L-amino acids, in particular L-lysine, L-threonine and L-aspartic acid.

In order to achieve the object of the present invention, the present invention provides, in a first aspect, the use of NCBI reference sequences NCgl2493 and/or NCgl0224 having reduced or absent activity and/or expression in the fermentative production of L-amino acids in coryneform bacteria.

In a second aspect, the present invention provides a method for the fermentative production of an L-amino acid or a method for increasing the fermentative production of an L-amino acid, comprising:

(1) modifying genes encoding NCBI reference sequences NCgl2493 and/or NCgl0224 on a chromosome of the coryneform bacterium to reduce or eliminate the activity and/or the expression quantity of the NCgl2493 and/or the NCgl 0224;

(2) and (2) fermenting and producing the L-amino acid by using the bacteria transformed in the step (1).

Further, the modification in step (1) comprises subjecting the gene encoding NCBI reference sequences NCgl2493 and/or NCgl0224 on a chromosome of a coryneform bacterium to substitution, deletion, and/or addition of one or more nucleotides.

Further, the modification in step (1) comprises knockout of a gene encoding NCBI reference sequences NCgl2493 and/or NCgl0224 on a chromosome of a coryneform bacterium.

In a third aspect, the invention provides an engineering bacterium for high yield of L-amino acid, wherein the engineering bacterium is a gene weakening strain obtained by weakening genes encoding NCBI reference sequences NCgl2493 and/or NCgl0224 in an original strain; or

The engineering bacteria are gene weakening strains obtained by weakening genes encoding NCBI reference sequences NCgl2493 and/or NCgl0224 in original strains and weakening genes related to an L-amino acid metabolic pathway; or

Enhancing genes related to the L-amino acid biosynthesis pathway and/or genes related to feedback inhibition desensitization in the gene weakening strain to obtain the gene enhanced strain.

In the present invention, the attenuation includes knocking out or reducing the expression of the gene.

The original strain is a coryneform bacterium which produces an L-amino acid.

In the present invention, the coryneform bacterium is a strain belonging to the genus Corynebacterium (Corynebacterium). Or Brevibacterium (Brevibacterium)

Preference is given to Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes), Brevibacterium flavum (Brevibacterium flavum), Brevibacterium lactofermentum (Brevibacterium lactofermentum) or Corynebacterium ammoniagenes (Corynebacterium ammoniagenes).

More preferably, the original strain is Corynebacterium glutamicum (Corynebacterium glutamicum) with the preservation number of CGMCC No. 11942.

The amino acid sequences of the proteins encoded by the genes NCgl2493 and NCgl0224 are shown in SEQ ID NO.1 and SEQ ID NO. 3, respectively.

In the present invention, the L-amino acid is selected from the group consisting of L-lysine, L-threonine and L-aspartic acid, preferably L-lysine.

In a fourth aspect, the present invention provides a method for constructing an engineered bacterium with high L-amino acid yield, comprising: genes encoding the NCBI reference sequences NCgl2493 and/or NCgl0224 in coryneform L-amino acid-producing bacteria are attenuated by genetic engineering means.

Preferably, methods of attenuation include, but are not limited to, mutagenesis, site-directed mutagenesis, homologous recombination.

In one embodiment of the present invention, a method for constructing an engineered bacterium with high L-amino acid yield comprises: respectively amplifying upstream and downstream fragments of a base deletion site of a gene NCgl2493 by using C1 and C2, C3 and C4 as primers and using genomic DNA of Corynebacterium glutamicum ATCC13032 as a template; then, taking the mixture of the upstream fragment and the downstream fragment as a template, taking C1 and C4 as primers to perform overlap PCR amplification to obtain an NCgl2493 gene fragment deletion product, connecting the NCgl2493 gene deletion product with a pK18mobsacB vector to obtain a pK18 mobsacB-delta NCgl2493 recombinant plasmid, introducing the recombinant plasmid into corynebacterium glutamicum with the preservation number of CGMCC No.11942, and screening positive transformants which are named as MHZ-1.

In another embodiment of the present invention, a method for constructing an engineered bacterium with high L-amino acid productivity comprises: respectively amplifying upstream and downstream fragments of a base deletion site of NCgl0224 gene by using D1 and D2, D3 and D4 as primers and using genomic DNA of Corynebacterium glutamicum ATCC13032 as a template; then taking the mixture of the upstream fragment and the downstream fragment as a template, taking D1 and D4 as primers to perform overlap PCR amplification to obtain a NCgl0224 gene fragment deletion product, connecting the NCgl deletion product with a pK18mobsacB vector to obtain a pK18 mobsacB-delta NCgl0224 recombinant plasmid, introducing the recombinant plasmid into corynebacterium glutamicum with the preservation number of CGMCC No.11942, and screening positive transformants which are named as MHZ-2.

In another embodiment of the present invention, a method for constructing an engineered bacterium that produces L-amino acids at high yield comprises: the pK18 mobsacB-. DELTA.NCgl 0224 recombinant plasmid was introduced into MHZ-1, and a positive transformant was selected and named MHZ-3.

The primer sequences of C1-C4 are respectively shown as SEQ ID NO. 5-8. The primer sequences of D1-D4 are respectively shown as SEQ ID NO 9-12.

In a fifth aspect, the invention provides the use of the engineered bacteria or the engineered bacteria constructed according to the above method in the fermentative production of L-amino acids, in particular L-lysine.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the engineering bacteria for high yield of L-amino acid provided by the invention have higher L-amino acid (especially L-lysine) production capacity, and NCgl2493 gene and/or NCgl0224 gene expression protein in corynebacteria cells is inactivated or weakened. Experiments show that the engineering bacteria for high yield of L-amino acid is taken as a fermentation strain, the L-lysine yield and the conversion rate are obviously improved, a foundation is laid for the industrial production of L-lysine, and the application prospect is wide. In addition, the method has no conflict with the existing modified chromosome modification sites of a large amount of bacteria with high yield of L-aspartic acid family amino acid, and can superpose the improvement effect, thereby being practically used for producing L-amino acid by fermenting various bacteria.

Drawings

FIG. 1 is a simplified structural diagram of recombinant plasmid pK18 mobsacB-. DELTA.NCgl 2493 of the present invention.

FIG. 2 is a simplified structural diagram of recombinant plasmid pK18 mobsacB-. DELTA.NCgl 0224 of the present invention.

Detailed Description

The present invention provides a coryneform bacterium which produces L-amino acid at a high yield, has L-amino acid-producing ability, and has inactivated or attenuated intracellular NCgl2493 gene and/or NCgl0224 gene-expressed protein.

In the invention, the gene inactivation or weakening mode modification is not limited to point mutation, knockout or promoter change and the like. The term "modification" means that the object to be modified is changed accordingly to achieve a certain effect. Means for engineering genes located on the chromosome include, but are not limited to, mutagenesis, site-directed mutagenesis or homologous recombination, preferably site-directed mutagenesis or homologous recombination. Modifying a gene located on a chromosome so that one or more nucleotides are added, deleted or substituted for the nucleotide sequence of the gene, for example, a nonsense codon may be inserted into the gene, or the gene may be knocked out. The gene may also be indirectly modified by modifying its regulatory sequence, so that the activity and/or expression level of the encoded protein is reduced.

In the present invention, the microorganism having L-lysine productivity can be selected from the group consisting of Corynebacterium glutamicum ATCC13032, Corynebacterium thermoaminogenes FERM BP-1539, Corynebacterium glutamicum KFCC10881 and Corynebacterium glutamicum KFCC11001, wherein the coryneform bacteria refer to microorganisms belonging to the genus Corynebacterium or Brevibacterium. Examples of coryneform bacteria that can be used in the present invention include, but are not limited to, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, and Corynebacterium ammoniagenes.

The inventors found some putative protein genes from the genome sequence database of the completely analyzed sequence of Corynebacterium glutamicum ATCC13032, and discovered that the content of intracellular L-aspartic acid and further the content of amino acids in the L-aspartate family, particularly L-lysine and L-threonine, can be increased by reducing the expression of NCgl2493 protein and NCgl0224 protein by chance.

In the present invention, the NCBI reference sequence NCgl2493 protein is a "hypothetical protein", the amino acid sequence of which is shown in SEQ ID NO:1, and the nucleotide sequence of the gene encoding NCgl2493 is shown in SEQ ID NO: 2. Although the specific activity of the NCgl2493 protein is unknown, in a specific embodiment of the present invention, the content of L-aspartic acid is increased and the yield of L-lysine and L-threonine is increased after the NCgl2493 gene is knocked out (i.e., its activity and/or expression amount is lost). Therefore, in the present invention, the activity and/or expression level of NCgl2493 preferably disappears.

In the present invention, the NCBI reference sequence NCgl0224 protein is a "hypothetical protein", the amino acid sequence of which is shown in SEQ ID NO. 3, and the nucleotide sequence of the gene encoding NCgl0224 is shown in SEQ ID NO. 4. Although the specific activity of NCgl0224 protein is unknown, in the specific embodiment of the present invention, after NCgl0224 gene is knocked out (i.e., its activity and/or expression amount disappears), the content of L-aspartic acid is increased, and the yield of L-lysine and L-threonine is increased. Therefore, in the present invention, the activity and/or expression level of NCgl0224 preferably disappears.

In some embodiments, the corynebacterium glutamicum has a deletion of a segment of the NCgl2493 gene encoding a mutant NCgl2493 protein group in its cell, i.e., the corynebacterium glutamicum has a loss of intracellular NCgl2493 protein activity, designated MHZ-1.

In some embodiments, the corynebacterium glutamicum has a deletion of a segment of the NCgl0224 gene encoding a mutant NCgl0224 protein group in its cells, i.e., the corynebacterium glutamicum has a loss of activity of the NCgl0224 protein in its cells, designated MHZ-2.

The invention also provides a construction method of the coryneform bacterium with high lysine yield, which comprises the steps of preparing a gene fragment deletion of a coding gene of a key enzyme, connecting the gene fragment deletion with a vector to obtain a recombinant vector, and transforming the coryneform bacterium to obtain the coryneform bacterium with high lysine yield.

In the invention, the vector is pK18mobsacB vector. In the present invention, the modification is preferably modification by homologous recombination, more preferably knockout by homologous recombination.

In some embodiments, the coryneform bacterium used in the method for constructing a coryneform bacterium having a high L-lysine productivity is Corynebacterium glutamicum having a accession number of CGMCC No.11942, see CN 105734004B. The strain has the capacity of producing L-lysine, and the NCgl2493 gene and the NCgl0224 gene are not optimized and modified. Competent cells of Corynebacterium glutamicum CGMCC No.11942 can be prepared by the skilled worker according to the classical method of cereal bars (C.glutamicum Handbook, Charpter 23).

The method for preparing the NCgl2493 gene fragment deletion recombinant vector specifically comprises the following steps: C1/C2 and C3/C4 are used as primers, the genomic DNA of Corynebacterium glutamicum ATCC13032 is used as a template, upstream and downstream fragments of a NCgl2493 base deletion site are respectively amplified, a mixture of the upstream and downstream fragments is used as a template, a primer group C1/C4 is used for performing overlap PCR amplification to obtain a NCgl2493 gene fragment deletion product, and the NCgl2493 gene fragment deletion product is connected with a pK18mobsacB vector to obtain a pK18 mobsacB-delta NCgl2493 recombinant plasmid.

The method for preparing the NCgl0224 gene fragment deletion recombinant vector specifically comprises the following steps: the upstream and downstream fragments of the NCgl0224 base deletion site were amplified respectively using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and the mixture of the upstream and downstream fragments as a template by primers D1/D2 and D3/D4, and the deletion product of the NCgl0224 gene fragment was obtained by overlap PCR amplification using primer set D1/D4, and ligated with pK18mobsacB vector to obtain pK18mobsacB- Δ NCgl0224 recombinant plasmid.

Wherein the sequences of the primers C1-C4 are respectively shown as SEQ ID NO. 5-8. The sequences of the primers D1-D4 are respectively shown as SEQ ID NO 9-12.

The corynebacterium glutamicum engineering bacteria constructed by the invention can be used for fermentation production, can realize effective accumulation of L-amino acid in the fermentation process, and has higher conversion rate. The invention therefore also provides the use of the Corynebacterium glutamicum for the fermentative production of L-lysine.

Furthermore, the invention also provides a production method of L-lysine, which comprises the step of inoculating the coryneform bacterium (corynebacterium glutamicum engineering bacterium) with high lysine yield into a fermentation culture medium for fermentation culture.

The fermentation medium used in the present invention had the following composition: glucose 60g/L, (NH)₄)₂SO₄ 25g/L，KH₂PO₄2.0g/L，MgSO₄·7H₂O1.0 g/L, yeast powder 10g/L, CaCO₃ 30g/L，pH7.0。

Preferably, the fermentation culture conditions are: shaking culture at 33 deg.C, rotating speed of 200rpm, and fermentation period of 15 h.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

The pK18mobsacB vector used in the following examples is a universal vector, which is a premium from the institute for microorganisms of the national academy of sciences.

Example 1 construction of recombinant plasmids pK18 mobsacB-. DELTA.NClg 2493, pK18 mobsacB-. DELTA.NCgl 0224

The nucleotide sequence of Corynebacterium glutamicum ATCC13032 NCgl2493 gene was obtained in GenBank database, and it was designed to introduce base deletion at a specific position (knockout of ORF sequence of the gene) to inactivate NCgl2493 gene, and four primers C1 to C4(SEQ ID NO:5-8) were synthesized based on the base sequence and the selected deletion position. PCR was carried out using ATCC13032 genome as a template and C1/C2 and C3/C4 as primers to obtain upstream and downstream homologous arm fragments, respectively, PCR was carried out using a mixture of the two fragments as a template using a primer set C1/C4 to obtain a full-length fragment, digestion was carried out using BamHI/HindIII, and at the same time, digestion was carried out using BamHI/HindIII on pK18mobsacB, and the fragment was ligated to a vector using T4DNA ligase to transform a Trans1T1 competent cell to obtain a recombinant plasmid pK18 mobsacB-. DELTA.NCgl 2493.

The construction of pK18 mobsacB-delta NCgl0224 plasmid is similar to that described above, the 225bp nucleotide sequence from 301 to 525 th of ORF sequence of NCgl0024 gene is knocked out, the used primers are D1/D2(SEQ ID NO:9-10), D3/D4(SEQ ID NO:11-12) PCR amplification upstream and downstream homologous arm fragments, and D1/D4 primer PCR amplification is carried out to obtain full-length product, finally pK18 mobsacB-delta NCgl0224 recombinant plasmid is obtained.

The simplified structural schematic diagrams of the recombinant plasmids pK18 mobsacB-delta NCgl2493 and pK18 mobsacB-delta NCgl0224 are shown in FIGS. 1 and 2, respectively.

Example 2 construction of NCgl2493 protein-inactivating Strain

CGMCC No.11942 competent cells were prepared according to the classical method of bars. The recombinant plasmid pK18 mobsacB-. DELTA.NCgl 2493 was used to transform the competent cells by electroporation, and transformants were selected on a selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the culture temperature of 33 ℃ under the shaking culture of a rotary table at 200 rpm. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted in gradient (10)^-2Continuously diluting to 10^-4) The diluted solution was applied to a common solid brain heart infusion medium containing 10% sucrose (commercial culture medium available from Obotaxacum Biotech, Inc., Beijing) and subjected to static culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry inserted vector sequences in their genome. The objective mutant strain is obtained by PCR amplification of the objective sequence and nucleotide sequencing analysis and is named MHZ-1.

EXAMPLE 3 construction of NCgl0224 protein-inactivated Strain

CGMCC No.11942 competent cells were prepared according to the classical method of bars. The competent cells were transformed with the recombinant plasmid pK18 mobsacB-. DELTA.NCgl 0224 by electroporation, and transformants in which the gene of interest was inserted into the chromosome due to homology were selected on a selection medium containing 15mg/L kanamycin. Culturing the screened transformant in a common liquid brain heart infusion culture medium overnightThe culture temperature was 33 ℃ and shaking culture was carried out on a rotary shaker at 200 rpm. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted in gradient (10)^-2Continuously diluting to 10^-4) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10% of sucrose, and is subjected to static culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry inserted vector sequences in their genome. The objective mutant strain is obtained by PCR amplification of the objective sequence and nucleotide sequencing analysis and is named MHZ-2.

Example 4 construction of strains in which NCgl2493 and NCgl0224 proteins were simultaneously inactivated

MHZ-1 competent cells were prepared according to the classical method of cereal bars. The competent cells were transformed with the recombinant plasmid pK18 mobsacB-. DELTA.NCgl 0224 by electroporation, and transformants in which the gene of interest was inserted into the chromosome due to homology were selected on a selection medium containing 15mg/L kanamycin. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the culture temperature of 33 ℃ under the shaking culture of a rotary table at 200 rpm. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted in gradient (10)^-2Continuously diluting to 10^-4) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10% of sucrose, and is subjected to static culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry inserted vector sequences in their genome. The objective mutant strain is obtained by PCR amplification of the objective sequence and nucleotide sequencing analysis and is named MHZ-3.

EXAMPLE 5 fermentative production of L-amino acids

The genetically engineered strains constructed in examples 2-4 were subjected to fermentation culture in 500ml triangular shake flasks (liquid containing 20ml, inoculum size 20% v/v, seed liquid OD)₅₆₂10), shaking and culturing by using a reciprocating shaking table, rotating speed 200rpm, culturing temperature 33 ℃, culturing time 15 hours, and setting three parallels in each group of experiments. The fermentation medium comprises the following components: glucose 60g/L, (NH)₄)₂SO₄ 25g/L，KH₂PO₄ 2.0g/L，MgSO₄·7H₂O1.0 g/L, yeast powder 10g/L, CaCO₃30g/L, pH7.0 adjusted by NaOH. The results of the fermentation of each genetically engineered strain to produce L-lysine are shown in Table 1.

TABLE 1 results of L-lysine production by fermentation of genetically engineered strains

As can be seen from Table 1, the deletion of NCgl2493 gene inactivated NCgl2493 protein had an ability to increase L-lysine, which was significantly different (P < 0.05) from that of the control group, and the precursor L-aspartic acid and the by-product L-threonine were significantly increased (P < 0.05). The inactivation of the protein is helpful to improve the content of the L-aspartic acid, and further can improve the yield of the L-aspartic family amino acids such as L-lysine and L-threonine.

The NCgl0224 gene is knocked out, so that the NCgl0224 protein is inactivated, the yield of the L-lysine can be improved, and compared with a control group, the yield of the L-lysine is obviously different (P < 0.05), and the precursor L-aspartic acid and the byproduct L-threonine are obviously improved (P < 0.05). The inactivation of the protein is helpful to improve the content of L-aspartic acid, and further can improve the yield of L-aspartic family amino acids, such as L-lysine and L-threonine.

The NCgl2493 and NCgl0224 genes are knocked out simultaneously, so that the NCgl2493 and NCgl0224 proteins are inactivated, the yield of L-lysine can be improved to the maximum extent, and compared with a control group, the L-lysine has obvious difference (P < 0.05), and the precursor L-aspartic acid and the byproduct L-threonine are obviously improved (P < 0.05). The inactivation of the two proteins is helpful to improve the content of L-aspartic acid, and further can improve the yield of amino acids in the L-aspartic family, such as L-lysine and L-threonine.

In conclusion, the downregulation of the expression of the NCgl2493 and/or NCgl0224 genes in coryneform bacteria contributes to the improvement of the L-lysine and L-threonine production, which is maximized if the expression of the NCgl2493 and/or NCgl0224 genes is simultaneously downregulated. Compared with the control, the content of L-aspartic acid is increased, and the yield of amino acids in the L-aspartic acid family, such as L-lysine, L-threonine and the like, can be further increased. Can be applied to producing bacteria for producing L-lysine or L-threonine, and has wide application prospect.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

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aaaacgccca catcaccgcg gcggcgaaat 30

<210> 11

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aaaacgccca catcaccgcg gcggcgaaat 30

<210> 12

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cacaagcttc ggggtctcga accacgtca 29

Claims

Use of NCBI reference sequences NCgl2493 and/or NCgl0224 having reduced or absent activity and/or expression levels in the fermentative production of L-amino acids in coryneform bacteria.
2. A method for producing an L-amino acid by fermentation or a method for increasing the fermentation yield of an L-amino acid, comprising:

(1) modifying genes encoding NCBI reference sequences NCgl2493 and/or NCgl0224 on a chromosome of the coryneform bacterium to reduce or eliminate the activity and/or the expression quantity of the NCgl2493 and/or the NCgl 0224;

(2) and (2) fermenting and producing the L-amino acid by using the bacteria transformed in the step (1).
3. The method according to claim 2, wherein the modification in step (1) comprises one or more nucleotide substitutions, deletions and/or additions to the gene encoding NCBI reference sequence NCgl2493 and/or NCgl0224 on the chromosome of the coryneform bacterium; or

The modification in step (1) comprises knockout of a gene encoding NCBI reference sequences NCgl2493 and/or NCgl0224 on a chromosome of a coryneform bacterium.
4. An engineering bacterium for producing L-amino acid with high yield, which is a gene weakening strain obtained by weakening the gene of a coding NCBI reference sequence NCgl2493 and/or NCgl0224 in an original strain; or

The engineering bacteria are gene weakening strains obtained by weakening genes encoding NCBI reference sequences NCgl2493 and/or NCgl0224 in original strains and weakening genes related to an L-amino acid metabolic pathway; or

Enhancing genes related to an L-amino acid biosynthesis pathway and/or genes related to feedback inhibition desensitization in the gene attenuation strain to obtain a gene enhancement strain;

said attenuation comprises knocking out or reducing expression of the gene;

the original strain is a coryneform bacterium which produces an L-amino acid.
5. The engineered bacterium of claim 4, wherein the coryneform bacterium is a strain of the genus Corynebacterium (Corynebacterium) or Brevibacterium (Brevibacterium);

preference is given to Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes), Brevibacterium flavum (Brevibacterium flavum), Brevibacterium lactofermentum (Brevibacterium lactofermentum) or Corynebacterium ammoniagenes (Corynebacterium ammoniagenes).
6. The engineered bacterium of claim 4, wherein the original strain is Corynebacterium glutamicum (Corynebacterium glutamicum) with a collection number of CGMCC No. 11942.
7. The engineered bacterium of any one of claims 4 to 6, wherein said L-amino acid is selected from the group consisting of L-lysine, L-threonine, and L-aspartic acid.
8. The construction method of the engineering bacteria for high yield of the L-amino acid is characterized by comprising the following steps: attenuating genes encoding NCBI reference sequences NCgl2493 and/or NCgl0224 in coryneform L-amino acid-producing bacteria by genetic engineering means;

said attenuation comprises knocking out or reducing expression of the gene;

the corynebacteria are strains in Corynebacterium (Corynebacterium) or Brevibacterium (Brevibacterium);

preferably, the coryneform bacterium is Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes), Brevibacterium flavum (Brevibacterium lactofermentum), Brevibacterium lactofermentum (Corynebacterium ammoniagenes), or Corynebacterium ammoniagenes (Corynebacterium ammoniagenes).
9. The method of claim 8, wherein the attenuation is performed by at least one method selected from the group consisting of mutagenesis, site-directed mutagenesis, and homologous recombination.
10. Use of the engineered bacterium of any one of claims 4 to 7 or constructed according to the method of claim 8 or 9 for the fermentative production of L-amino acids;

preferably, the L-amino acid is selected from the group consisting of L-lysine, L-threonine, and L-aspartic acid.